Identification and Application of Two Promising Peptide Ligands for the Immunodetection of Imidacloprid Residue.

As the most widely used neonicotinoid insecticide, it is of great significance to explore the immunoreagents and immunoassays for imidacloprid (IMI) residue. In immunoassays, specific peptide ligands, such as peptidomimetic and anti-immunocomplex peptides, are regarded as promising substitutes for chemical haptens. In the present work, we identified thirty sequences of peptidomimetics and two sequences of anti-immunocomplex peptides for IMI from three phage pVIII display cyclic peptide libraries, in which the anti-immunocomplex peptides are the first reported noncompetitive reagents for IMI. The peptidomimetic 1-9-H and anti-immunocomplex peptide 2-1-H that showed the best sensitivity were utilized to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), with a half inhibition concentration of 0.55 ng/mL for competitive P-ELISA and a half-saturation concentration of 0.35 ng/mL for noncompetitive P-ELISA. The anti-immunocomplex peptide was demonstrated to greatly improve the specificity compared with competitive P-ELISA. In addition, the accuracy of proposed P-ELISAs was confirmed by recovery analysis and HPLC verification in agricultural and environmental samples. These results show that the peptide ligands identified from phage display library can replace chemical haptens in the immunoassays of IMI with the satisfactory performance.

Development of competitive and noncompetitive immunoassays for clothianidin with high sensitivity and specificity using phage-displayed peptides.

Clothianidin is a widely used pesticide that has been banned from outdoor use by the European Union due to its toxicity. To improve the sensitivity and specificity of existing clothianidin immunoassays, we developed competitive and noncompetitive immunoassays for clothianidin based on phage-displayed peptides. Cyclic 8-, 9-, and 10-residue peptide libraries were constructed using an optimized phagemid pComb-pVIII to prevent the loss of theoretical library diversity. Twenty-eight peptidomimetics and two anti-immunocomplex peptides were isolated through a blended panning process and used to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), respectively. After optimization, the half inhibition concentration (IC50) and half saturation concentration (SC50) of competitive and noncompetitive P-ELISAs were 3.83 ± 0.23 and 0.45 ± 0.02 ng/mL, respectively. Competitive P-ELISA showed 2.6-18.2% cross-reactivity with imidaclothiz, nitenpyram and imidacloprid. Importantly, noncompetitive P-ELISA, which has the best specificity and great sensitivity for clothianidin, showed no cross-reactivity with the analogs. The average recoveries of competitive and noncompetitive P-ELISAs were 73.8-104.1% and 76.6-102.2%, respectively, while the relative standard deviations were ≤ 11.0%. In addition, the results of P-ELISAs in the analysis of blind samples were consistent with those of high-performance liquid chromatography.

Competitive and noncompetitive fluorescence polarization immunoassays for the detection of benzothiostrobin using FITC-labeled dendrimer-like peptides.

Peptides obtained from phage display libraries are valuable reagents for small-molecule immunoassays. However, their application in fluorescence polarization immunoassays (FPIAs) is limited by phage particles. Here, monomer, dendrimer-like dimer, tetramer peptidomimetic and anti-immunocomplex tracers were designed and synthesized using lysine as special scaffolds and spacers to develop competitive and noncompetitive FPIAs for benzothiostrobin. The affinity between tracers and monoclonal antibodies or immunocomplexes increased with the tracer valence. A higher signal-to-noise ratio and sensitivity could be generated in the FPIAs based on tetramer tracers. The sensitivities of competitive (50% inhibitory concentration) and noncompetitive (50% saturation concentration) FPIAs were 19.71 ± 4.65 and 40.43 ± 2.73 ng mL-1, respectively. The spiked recoveries were 78.3%-105.2% with relative standard deviations (RSDs) of 0.7%-15.4% for the competitive FPIA, while 78.7%-115.3% with RSDs of 0.7%-12.5% for the noncompetitive FPIA. The amounts of benzothiostrobin in rice detected by the FPIAs were consistent with those detected by high performance liquid chromatography.